The long-term goal of this proposal is to understand the molecular basis for lymphocyte anergy and tolerance. Its specific objective is to investigate the role of NFAT, a Ca2+-activated transcription factor, in these processes. We have shown that NFAT can turn on two opposing gene expression programmes in T cells: a programme of productive activation defined by upregulation of genes encoding cytokines, chemokines and cell surface receptors, whose regulatory regions typically contain composite binding sites for NFAT and its transcriptional partner AP-1 (Fos-Jun); and a non-overlapping programme of negative regulation, induced by NFAT in the absence of AP-1, whose functional consequence is to render T cells "anergic", i.e. unresponsive to subsequent stimulation through the TCP. This application will follow up on these earlier findings. Aim 1 is to systematically identify potential targets and effectors of the Ca2+-induced negative regulatory programme, and test whether they affect T cell responses or influence anergy induction. Aim 2 is to investigate the relation between NFAT and regulatory T cells. In collaboration with a structural laboratory, we have shown that FoxP proteins bind cooperatively with NFAT on composite NFAT: AP-1 sites. We will examine the role of this interaction in the function of regulatory T cells. Aim 3 is to define the contribution of NFAT, its AP-1 and FoxP partners and its target/ effector proteins in a selected model of autoimmunity in mice. These studies are important from a clinical perspective: being able to manipulate T cell tolerance is valuable in almost every immunological setting including transplant medicine, asthma and allergy, and diverse manifestations of autoimmune disease.